Affiliation: Department of Pathology, United States Military Cancer Institute, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA. t-galbaugh@northwestern.edu

ABSTRACT

Background: HC11 mouse mammary epithelial cells differentiate in response to lactogenic hormone resulting in expression of milk proteins including beta-casein. Previous studies have shown that epidermal growth factor (EGF) blocks differentiation not only through activation of the Ras/Mek/Erk pathway but also implicated phosphatidylinositol-3-kinase (PI-3-kinase) signaling. The current study analyzes the mechanism of the PI-3-kinase pathway in an EGF-induced block of HC11 lactogenic differentiation.

Results: HC11 and HC11-luci cells, which contain luciferase gene under the control of a beta-casein promotor, were treated with specific chemical inhibitors of signal transduction pathways or transiently infected/transfected with vectors encoding dominant negative-Akt (DN-Akt) or conditionally active-Akt (CA-Akt). The expression of CA-Akt inhibited lactogenic differentiation of HC11 cells, and the infection with DN-Akt adenovirus enhanced beta-casein transcription and rescued beta-casein promotor-regulated luciferase activity in the presence of EGF. Treatment of cells with Rapamycin, an inhibitor of mTOR, blocked the effects of EGF on beta-casein promotor driven luciferase activity as effectively as PI-3-kinase inhibitors. While expression of CA-Akt caused a constitutive activation of p70S6 kinase (p70S6K) in HC11 cells, the inhibition of either PI-3-kinase or mTOR abolished the activation of p70S6K by EGF. The activation of p70S6K by insulin or EGF resulted in the phosphorylation of ribosomal protein S6 (RPS6), elongation initiation factor 4E (elF4E) and 4E binding protein1 (4E-BP1). But lower levels of PI-3-K and mTOR inhibitors were required to block insulin-induced phosphorylation of RPS6 than EGF-induced phosphorylation, and insulin-induced phosphorylation of elF4E and 4E-BP1 was not completely mTOR dependent suggesting some diversity of signaling for EGF and insulin. In HC11 cells undergoing lactogenic differentiation the phosphorylation of p70S6K completely diminished by 12 hours, and this was partly attributable to dexamethasone, a component of lactogenic hormone mix. However, p70S6K phosphorylation persisted in the presence of lactogenic hormone and EGF, but the activation could be blocked by a PI-3-kinase inhibitor.

Figure 1: The effect of the PI-3-kinase inhibitor, LY294002 on epidermal growth factor (EGF) disruption of differentiation. A. HC11 cells were induced to differentiate in DIP-induction media with and without EGF (10 ng/ml) and LY294002 (5 μM) for 48 hours. β-casein induction was determined via northern blot and was normalized to β-actin. B. HC11 cells were grown to confluence as stated above and induced to differentiate in DIP-induction media with and without EGF (10 ng/ml) and LY294002 (10 μM). Cells were photographed at 96 hours post-induction. The number of mammospheres per field is reported: this was determined by counting the number of mammospheres per low power field and determining the mean of five fields.

Mentions:
EGF stimulation of HC11 cells activates PI-3-kinase signaling as well as other pathways, and the results from our previous study determined that EGF blocked activation of a β-casein promotor-luciferase activity following induction of lactogenic differentiation via both Mek/Erk and PI-3-kinase dependent mechanisms [6]. The results in figure 1 confirm and expand those findings using an inhibitor of PI-3-kinase activity. β-casein RNA transcription was examined by northern blotting following stimulation of HC11 cells with the lactogenic hormone mix, DIP, in the presence and absence of EGF and LY294002. EGF blocked lactogenic hormone induced β-casein transcription and the addition of the PI-3-kinase inhibitor, LY294002, partially rescued β-casein transcription (figure 1A). However, the addition of PI-3-kinase inhibitors LY294002 or wortmannin in the absence of EGF reduced all markers of lactogenic differentiation (data not shown), indicating that survival signaling from this pathway was essential for HC11 differentiation to proceed.

Figure 1: The effect of the PI-3-kinase inhibitor, LY294002 on epidermal growth factor (EGF) disruption of differentiation. A. HC11 cells were induced to differentiate in DIP-induction media with and without EGF (10 ng/ml) and LY294002 (5 μM) for 48 hours. β-casein induction was determined via northern blot and was normalized to β-actin. B. HC11 cells were grown to confluence as stated above and induced to differentiate in DIP-induction media with and without EGF (10 ng/ml) and LY294002 (10 μM). Cells were photographed at 96 hours post-induction. The number of mammospheres per field is reported: this was determined by counting the number of mammospheres per low power field and determining the mean of five fields.

Mentions:
EGF stimulation of HC11 cells activates PI-3-kinase signaling as well as other pathways, and the results from our previous study determined that EGF blocked activation of a β-casein promotor-luciferase activity following induction of lactogenic differentiation via both Mek/Erk and PI-3-kinase dependent mechanisms [6]. The results in figure 1 confirm and expand those findings using an inhibitor of PI-3-kinase activity. β-casein RNA transcription was examined by northern blotting following stimulation of HC11 cells with the lactogenic hormone mix, DIP, in the presence and absence of EGF and LY294002. EGF blocked lactogenic hormone induced β-casein transcription and the addition of the PI-3-kinase inhibitor, LY294002, partially rescued β-casein transcription (figure 1A). However, the addition of PI-3-kinase inhibitors LY294002 or wortmannin in the absence of EGF reduced all markers of lactogenic differentiation (data not shown), indicating that survival signaling from this pathway was essential for HC11 differentiation to proceed.

Affiliation:
Department of Pathology, United States Military Cancer Institute, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA. t-galbaugh@northwestern.edu

ABSTRACT

Background: HC11 mouse mammary epithelial cells differentiate in response to lactogenic hormone resulting in expression of milk proteins including beta-casein. Previous studies have shown that epidermal growth factor (EGF) blocks differentiation not only through activation of the Ras/Mek/Erk pathway but also implicated phosphatidylinositol-3-kinase (PI-3-kinase) signaling. The current study analyzes the mechanism of the PI-3-kinase pathway in an EGF-induced block of HC11 lactogenic differentiation.

Results: HC11 and HC11-luci cells, which contain luciferase gene under the control of a beta-casein promotor, were treated with specific chemical inhibitors of signal transduction pathways or transiently infected/transfected with vectors encoding dominant negative-Akt (DN-Akt) or conditionally active-Akt (CA-Akt). The expression of CA-Akt inhibited lactogenic differentiation of HC11 cells, and the infection with DN-Akt adenovirus enhanced beta-casein transcription and rescued beta-casein promotor-regulated luciferase activity in the presence of EGF. Treatment of cells with Rapamycin, an inhibitor of mTOR, blocked the effects of EGF on beta-casein promotor driven luciferase activity as effectively as PI-3-kinase inhibitors. While expression of CA-Akt caused a constitutive activation of p70S6 kinase (p70S6K) in HC11 cells, the inhibition of either PI-3-kinase or mTOR abolished the activation of p70S6K by EGF. The activation of p70S6K by insulin or EGF resulted in the phosphorylation of ribosomal protein S6 (RPS6), elongation initiation factor 4E (elF4E) and 4E binding protein1 (4E-BP1). But lower levels of PI-3-K and mTOR inhibitors were required to block insulin-induced phosphorylation of RPS6 than EGF-induced phosphorylation, and insulin-induced phosphorylation of elF4E and 4E-BP1 was not completely mTOR dependent suggesting some diversity of signaling for EGF and insulin. In HC11 cells undergoing lactogenic differentiation the phosphorylation of p70S6K completely diminished by 12 hours, and this was partly attributable to dexamethasone, a component of lactogenic hormone mix. However, p70S6K phosphorylation persisted in the presence of lactogenic hormone and EGF, but the activation could be blocked by a PI-3-kinase inhibitor.